Human placenta-derived mesenchymal stem cells stimulate neuronal regeneration by promoting axon growth and restoring neuronal activity

In the last decades, mesenchymal stem cells (MSCs) have become the cornerstone of cellular therapy due to their unique characteristics. Specifically human placenta-derived mesenchymal stem cells (hPMSCs) are highlighted for their unique features, including ease to isolate, non-invasive techniques for large scale cell production, significant immunomodulatory capacity, and a high ability to migrate to injuries. Researchers are exploring innovative techniques to overcome the low regenerative capacity of Central Nervous System (CNS) neurons, with one promising avenue being the development of tailored mesenchymal stem cell therapies capable of promoting neural repair and recovery. In this context, we have evaluated hPMSCs as candidates for CNS lesion regeneration using a skillful co-culture model system. Indeed, we have demonstrated the hPMSCs ability to stimulate damaged rat-retina neurons regeneration by promoting axon growth and restoring neuronal activity both under normoxia and hypoxia conditions. With our model we have obtained neuronal regeneration values of 10%–14% and axonal length per neuron rates of 19-26, μm/neuron. To assess whether the regenerative capabilities of hPMSCs are contact-dependent effects or it is mediated through paracrine mechanisms, we carried out transwell co-culture and conditioned medium experiments confirming the role of secreted factors in axonal regeneration. It was found that hPMSCs produce brain derived, neurotrophic factor (BDNF), nerve-growth factor (NGF) and Neurotrophin-3 (NT-3), involved in the process of neuronal regeneration and restoration of the physiological activity of neurons. In effect, we confirmed the success of our treatment using the patch clamp technique to study ionic currents in individual isolated living cells demonstrating that in our model the regenerated neurons are electrophysiologically active, firing action potentials. The outcomes of our neuronal regeneration studies, combined with the axon-regenerating capabilities exhibited by mesenchymal stem cells derived from the placenta, present a hopeful outlook for the potential therapeutic application of hPMSCs in the treatment of neurological disorders.


INTRODUCTION
Since the classic studies of Ramón y Cajal, the low regenerative capacity of the neurons of the Central Nervous System (CNS)(1) has been known.Since then, various strategies have been used to achieve neuronal regeneration, such as blocking axonal growth inhibitors or with cell therapy, using transplants of different cell types (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).For in vivo or future clinical studies, it would be essential to determine in vitro the neuroregenerative capacity of the cell populations that would be used in cell therapy10/26/23 2:10:00 PM.One of the best models to study in vitro and quantify cell-induced adult axonal regeneration is the co-culture of adult axotomized retinal ganglion cells (RGCs) with cells putatively capable of inducing axonal regeneration (olfactory ensheathing glia cells -OEGs, Schwann cells, astrocytes, etc.) (19)(20)(21)(22).Our group has carried out studies in cocultures of RGCs with OEG populations that have made possible to advance in the characterization of the molecular bases of the OEG-dependent regenerative capacity.We have shown that the ability of these cells to induce adult axonal regeneration in coculture depends on several molecules (7,17,18): they secrete neurotrophic factors (23)(24)(25), they produce extracellular matrix proteases that contribute to degrading the perineuronal network that stabilizes the environment of adult neurons (26); produce proteases that stimulate axon regeneration (27), etc.
Stem cells are unspecialized cell precursors that are able to self-renew and differentiate into one or more specialized cell types in response to specific signals (28)(29)(30)(31).In higher animals and based on their source, stem cells have been classified into two groups: on the one hand, embryonic stem cells (EScells), pluripotent, derived from the internal cell mass of the embryo at the blastocyst stage (7-14 days), that can generate all the different cell types in the body (32) and on the other hand, the organ-specific stem cells, derived from those, after many cell divisions, and which are multipotential; that is, they can originate the cells of a specific organ in the embryo, and also in the adult (33)(34)(35)(36).
It has been shown, using animal models of spinal cord injury (SCI), that different stem cells, both embryonic and adult, undifferentiated or differentiated in culture with different protocols, are capable of promoting neuro-regeneration after injury, as well as functional recovery (37)(38)(39).
The regenerative potential of mesenchymal stem cells of different origins has been studied and contrasted by numerous research groups (40)(41)(42).The source from which organ-specific mesenchymal stem cells have traditionally been extracted is bone marrow.These cells can generate all cell types of the blood and the immune system (35), they can be grown both in vitro in the laboratory and in vivo (using animal models) and have been tested in tissue repair experiments (43,44).Transplantation of bone marrow-derived cells into SCI models has been reported to promote axonal regeneration, reduce lesion size, and improve functional outcome (45)(46)(47)(48)(49)(50)(51)(52)(53).The precise cell type within bone marrow responsible for these beneficial effects is not fully established but is thought to reside within the marrow stromal, corresponding to the MSC population (54,55).Additionally, transplantation after SCI of bone marrow MSCs gene-modified to secrete brain BDNF give way to increased corticospinal neurons survival in primary motor cortex as compared with the unmodified MSCs and promoted effects in locomotor recovery not observed with the control MSCs (56).
However, the use of bone marrow stem cells has some limitations due to the invasiveness of the extraction method and the decrease in their proliferation and differentiation capacity with the maintenance of cells in culture (30,57).Many researchers are working on finding an alternative to these cells that can be used in clinical applications.Placental tissue is a source of cells of great value in regenerative medicine because hPMSCs are easily isolated and can be expanded in culture using a suitable medium, they have great phenotypic plasticity, and in addition, due to the fact that the placenta is involved in the maintenance of fetal tolerance during pregnancy, they develop immunomodulatory properties of great importance in clinical applications based on cell therapy.These characteristics point to hPMSCs being suitable candidates for use in cell therapies for CNS injury (58)(59)(60)(61).
For all these reasons, in this work we have, determined and quantified the ability of hPMSCs to induce adult axonal regeneration by using our model of co-culture with adult axotomized NGRs, under normoxic and hypoxic conditions.It is described that certain features of hPMSCs are stimulated when subjected to low oxygen concentrations in culture, including proliferation, migration, and neuroprotective potential (62)(63)(64)(65)(66)(67)(68).Furthermore, we understand that hypoxic conditions are representative of the physiological reality in some time course events after a CNS trauma or some neurological pathologies, such as a ischemic stroke.Therefore, we considered performing experiments simultaneously under normoxia and hypoxia conditions, which would allow us to compare the results in both environments.
Moreover, to assess whether the regenerative capabilities of hPMSCs are entirely contact-dependent effects or they are also mediated through paracrine mechanisms, we carried out Transwell co-culture and conditioned medium experiments confirming the role of secreted factors in RGCs' axonal regeneration.
Finally, through the application of the patch clamp technique, we assessed the functional recovery of regenerated neurons, thus confirming their electrophysiological activity.This provides valuable insights into the realm of neural regeneration and its potential implications for treating neurological disorders.

Proliferation capacity of hPMSCs
To examine the proliferation capacity of the hPMSCs in vitro, the cumulative population doubling (PD) was calculated over 30 days (from passage 3 to 8).The hPMSCs were placed in triplicate into 10 cm 2 multiwell dishes at a concentration of 10 4 cells/cm 2 and subcultured after 5 days at the same density.The cells were counted using a hemocytometer.The cumulative cell doubling of the cell populations was plotted against time in the culture to determine the growth kinetics of hPMSCs expansion.The number of population doubling was determined by counting the number of adherent cells at the start and end of each passage.The population doubling was calculated at every passage according to the equation: log2 (number of harvested cells/number of seeded cells).The finite population doubling was determined by the cumulative addition of the total numbers generated from each passage until the cells stopped dividing.

Mesodermal differentiation of hPMSCs
For adipogenic differentiation hPMSCs were seeded at an inoculation density of 2.4x10 5 cells/well in 6-well plates until they reached confluence and were then induced by three cycles of induction/maintenance with Differentiation media BulletKits -adipogenic (Lonza) according to the manufacturer's instructions.Adipogenesis was assayed by staining of intracellular lipid droplets with Oil Red O (Sigma, St. Louis, Missouri, USA).The monolayer cultures were incubated with Oil Red O solution for 10 min at ambient temperature and examined by microscopy.
For osteogenic differentiation hPMSCs were seeded at an inoculation density of 3x10 4 cells/well in 6-well plates and cultured in osteogenic induction medium Differentiation media BulletKit™ -osteogenic (Lonza) according to the manufacturer's instructions.After 21 days in osteogenic induction medium, mineral deposits were observed by Alizarin Red (Sigma) staining.For Alizarin Red staining, the cells were fixed with 10% formalin for 5 min.The formalin was removed, and cells were washed twice with water.Then, the cells were incubated with Alizarin Red S staining solution (1.4%, pH 4.0; Sigma) for 20 min at ambient temperature and examined by microscopy.
For chondrogenic differentiation micromass cultures were generated by seeding 5 µL droplets of 1.6x10 7 cells/mL cell solution in the center of multi-well plate wells.After cultivating micromass cultures for 2 hours StemPro® Chrondrogenesis differentiation medium (Gibco) was added.After 14 days of incubation, the micromasses were rinsed with PBS and fixed with 4% paraformaldehyde solution for 30 minutes.Chondrogenic differentiation was observed by Alcian Blue staining (Merck KGaA, Darmstadt, Germany) and examined by microscopy.

Quantitative polymerase chain reaction (qPCR) for pluripotent stem cell markers
Total RNA from hPMSCs was isolated using RNeasy Mini Kit (QIAGEN, Hilden, Germany).Reverse transcription (RT) reactions were carried out on 250 ng of total RNA using the High-Capacity cDNA Reverse Transcription Kit" (Applied Biosystems, Waltham, Massachusetts, USA).RT-PCR analysis for pluripotency markers genes such as OCT4, SOX y NANOG was carried out.SDS Software (Applied Biosystems) was used to analyze the data.

Flow cytometry analysis
To confirm MSCs phenotype of cells grown in normoxic and hypoxic conditions, cells were incubated with antibodies against the following human antigens: CD105, CD44, CD73, CD14, CD34, CD19, and CD45 and HLA-DR.After washing, cells were subjected to flow cytometry analysis using a flow cytometer (Miltenyi Biotech).The data were analyzed with MACSQuantify™ Software.

Axonal regeneration assays by indirect co-culture
Axotomized retinal neurons were plated onto 10 mg/ml PLL (Sigma) coated dishes and cultured in NB-B27 medium.hPMSCs were seeded directly onto 24-well 0.4 µm Transwell inserts at a density of 10 4 cells/cm 2 , cultured in NB-B27 medium and maintained in normoxia and hypoxia for 96 hours before fixing them with 4% PFA.

Axonal regeneration assays using conditioned media
hPMSCs were seeded at a density of 10 4 cells/cm 2 in HGCM.When cultures reached approximately 90% confluence, hPMSCs were rinsed with PBS (PBS EDTA pH 7.5; Lonza) and maintained in NB-B27 medium for 96 hours.Conditioned medium was collected and centrifuged at 1,200 g for 5 min.
Axotomized neurons were plated as described above.Conditioned medium or equivalent volume of NB-B27 medium (control) was added to the neurons.The cells were incubated for 96 hours and fixed with 4% PFA.
Axonal regeneration events were thoroughly characterized and quantified as described previously (21,22,26).Co-cultures were analyzed by immunocytochemistry employing an antibody against a phosphorylated form of MAP1B and Neurofilament-H (NF-H) axonal proteins (SMI31; Sternberger Monoclonal Inc., Baltimore, MD; 1:500) and an antibody, 514, which recognizes high molecular weight microtubule-associated protein 2 (MAP2A,B; 1:400) (70).The percentage of retinal neurons with an axon was determined by counting the number of MAP2A,B positive neurons that bear a polarized neurite that can be labelled with the antibody against the phosphorylated forms of MAP1B and NF-H proteins.The samples were observed in an inverted fluorescent microscope with a 40X immersion objective (DMi8, Leica, Wetzlar, Germany) and a minimum of 20 fields (containing a minimum of 200 neurons) were taken randomly.
Two parameters were determined: percentage of neurons that have regenerated axons (number of neurons with an axon versus total number of counted neurons), and the mean axonal length of the regenerated axons (µm)/neuron (ratio between the total length of the regenerated axons and the total number of counted neurons).Fluorescence signals were analyzed with ImageJ software (Image J, NIH) and the length of the axons was determined using the Neuron J plugin.

Immunocytochemistry
Direct and indirect co-cultures, and cultures with conditioned media, were fixed with 4% PFA to perform immunocytochemistry analysis.Samples were permeabilized and blocked in blocking solution (1X PBS, 0.1% triton, 1% FBS) for 30 minutes at room temperature.Next, they were incubated with the corresponding primary antibody in blocking solution at the optimal dilution for each of them.The primary antibodies used were SMI31 and 514.Incubation was carried out at 4 °C overnight.After washing, the cells were incubated for another hour in a solution of PBS-TS containing the appropriate fluorescent secondary antibody, at its optimal dilution.Fluorescence-conjugated secondary antibodies used were Alexa Fluor 488 and Alexa Fluor 594, respectively.
Finally, the samples were washed with PBS and mounted with Flouromount (Southern Biotech, Birmingham, Alabama, USA).The labelled preparations were visualized using an inverted with fluorescent microscope with a 40X immersion objective (DMi8, Leica).

Brain-Derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) quantification
BDNF and NGF levels in direct and indirect co-cultures, and in conditioned media, were evaluated using Human BDNF ELISA kit (Abcam, cat# ab1212166) and Human beta Nerve Growth Factor ELISA Kit (Abcam, cat# ab193760) following the manufacturer's instructions.

Electrophysiology
Electrophysiological recording of cell voltage and ionic currents were performed under the whole cell configuration of the platch clamp technique (Hamill, Marty, Neher, Sakmann, & Sigworth, 1981) using an Axopatch 200A amplifier (Axoninstruments, FosterCity, CA, USA).Borosilicate pipettes (1.2 mm outer diameter) with a tungsten internal filament, were made using a vertical pipette puller (Narishige mod.P88, Narishige, Tokyo, Japan).The inner diameter of the pipette after pulling was approximately 0.5 to 1 μm.Filling of the pipette was carried out with an intracellular solution containing (in mM): 10 NaCl, 110 KCl, 5 EGTA, 0.5 CaCl2, 1 MgCl2, and 10 glucose (pH 7.4).The electrical resistance of the pipettes was measured, in the range of 8 to 12 MΩ.The seal resistance was approximately 1 to 3 GΩ.Liquid junction potential was routinely corrected.The Ag-AgCl indifferent electrode was connected via an agarose bridge to the perfusion.The cell voltage was maintained at −80 mV and depolarizing pulses of 30 ms duration were applied in steps of 5 mV.Both the holding voltage and the pulses were generated using a personal computer connected to the CED plus (Cambridge Electronic Design Ltd, Cambridge, England).Data were sampled at 0.2-10 kHz after low-pass filtering with an appropriate cut-off for each sampling frequency.Data analysis was performed offline using a personal computer.

Statistical analysis
Data are presented as the mean ± standard deviation (SD).Multiple comparisons, oneway ANOVA followed by Dunnet´s post hoc test was used to evaluate the differences in axonal regeneration.An alpha value of p ≤ 0.05 was used for statistical significance.

Culture and characterization of hPMSCs
Prior to evaluate neural regeneration potential of hPMSCs, we confirmed that the cells were in agreement with the criteria of International Society for Cellular Therapy published in 2006 (71).
Mesenchymal cells attached to cell culture plastic flasks and showed a typical spindletype shape morphology when observed by inverted phase contrast microscopy (Figure 1A).No differences in morphology between oxygen conditions were detected.
The comparative growth kinetics of the hPMSCs grown in normoxia and hypoxia from passages 3 to 8 revealed that hypoxic cultures had a higher growth potential, measured as cumulative PDs (Figure 1B).
We then analyzed pluripotency markers expression (NANOG, Oct-4 and SOX2) and observed an increased relative expression when compared to the fibroblast cell lineage 293T (Figure 1C).
To assess the expression of mesenchymal and hematopoietic markers an immunophenotypic analysis was performed.Flow cytometry results indicated that hPMSCs expressed typical immunophenotypic characteristics consistent with those of a mesenchymal lineage.Cells cultured in both oxygen conditions were positive for surface markers CD105, CD44 and CD73 and negative for CD34, CD14, CD 45, CD 19, and HLA-DR (Figure 1D).
We investigated the differentiation potential of hPMSCs by inducing them into osteoblasts, adipocytes and chondrocytes in vitro.Alizarin red staining revealed significant calcium deposition in treated cells, thus confirming osteogenesis (Figure 1E), Oil Red O-stained lipid drops were observed in differentiated hPMSCs indicating adipogenesis (Figure 1F), and the presence of proteoglycan staining with Alcian blue proved chondrogenesis (Figure 1G).Interestingly, hPMSCs grown under hypoxic conditions showed a substantial increase in calcium deposition when compared to hPMSCs grown in normoxia.Similarly, hypoxic hPMSCs cultures showed a higher number of lipids droplets.These results suggest that hypoxia promotes hPMSCs differentiation potential.
Taken together, these results confirm that the cells of this study were mesenchymal stem cells.In addition, these data reveal that low oxygen concentration has no significant in vitro effect on the morphology and phenotype profiles of hPMSCs but it can promote both hPMSCs proliferation and differentiation abilities.

Neuroregenerative effect of hPMSCs
To evaluate hPMSCs neuroregenerative potential an in vitro regeneration assay was performed.All analyses were conducted on cells at passages 2-5, simultaneously cultured under normoxic and hypoxic conditions.Different densities of hPMSCs (5x10 4 , 8x10 4 and 10 5 ) were co-cultured with axotomized 2-month-old rat retinal ganglion cells (RGCs) in both normoxia and hypoxia, for 96 hours.In parallel, an immortalized human olfactory ensheathing cell line (TS12) was used as a low regenerative capacity control (69).Wells treated with the substrate PLL with no cells attached were used as a negative control for RGCs axonal regeneration capacity.Neuroregeneration was detected by immunofluorescence using SMI31 antibody (against MAP1B and-NF-H) as an axonal marker and 514 antibody (against MAP2A,B) to observe the somatodendritic compartment (Figure 2A).Two parameters were then quantified: the percentage of neurons with an axon, and the mean axonal length per neuron (µm/neuron).
Notably, these data demonstrate a high increment in axotomized RGCs regenerative capacity when cultured in the presence of hPMSCs.

Neurotrophic factors expression by hPMSCs
Western blot experiments confirmed pro-BDNF, pro-NT3 and pro-NGF synthesis by hPMSCs when grown both in HGCM or NB-B27 media (Figure 3A,B) at any time point studied.After quantification by Image J (data not shown) we concluded that pro-NGF synthesis by hPMSCs was 2 times higher when grown in hypoxia relative to normoxia, in both types of culture media.Cell growth in hypoxia triggers the expression of HIF as it is described (72).

Neuroregenerative effect of hPMSCs by indirect co-culture and conditioned medium collected from hPMSCs
Our previous results show that hPMSCs provided a permissive substrate that allowed axon regeneration and elongation in axotomized RGCs linked to neurotrophic pro-factors synthesized by hPMSCs.In order to evaluate the paracrine effect of neurotrophic factors secreted by hPMSCs two kind of in vitro indirect co-culture assays were performed.
For indirect co-culture assay: axotomized RGCs were plated onto PLL coated dishes.hPMSCs cell suspension (10 4 cells/cm 2 ) in NB-B27 medium was seeded onto the 0.4 µm Transwell inserts.These indirect co-cultures were performed in both normoxia and hypoxia.For secretome activity assay: NB-B27 medium was conditioned by hPMSCs in normoxia or hypoxia and later added to axotomized RGCs cultures on PLL (Figure 4A).Axonal regeneration was characterized and quantified as described previously (Figure 4B).
Results demonstrated the paracrine effect of hPMSCs on RGCs neuroregeneration.In indirect Transwell co-cultures, the percentage of RGCs with a regenerated axon is very similar to control cultures (PLL) in normoxia (7.41%±4.3)and hypoxia (6.19%±5.3).However, mean axonal length per neuron achieved is more than 2 times higher in both conditions (19.07±2.24mm/neuron in normoxia; 15.87±8.9mm /neuron in hypoxia).
Similar results were obtained when axotomized RGCs were cultured with hPMSCs CM.Under those conditions, although the percentage of RGC with a regenerated axon is similar to the control (PLL), the mean axonal length per neuron is higher in both normoxia and hypoxia.No differences were observed in the effect on regeneration when CM were conditioned in either normoxic or hypoxic conditions (Figure 4B).These results strongly suggest that hPMSCs function as a biological substrate that increase the capacity of RGCs to regenerate their axons, partly through a paracrine mechanism.

Study of functional properties of regenerated axons
After neuroregeneration potential of hPMSCs was confirmed, we further analyzed the developmental stage of the regenerated axons by the detection of mature synaptic vesicles and voltage-gated sodium channels (VGSCs).Additionally, we explore the axonal complete functionality by measuring their electrophysiological capacity.
SV2A is a synaptic vesicle membrane glycoprotein expressed exclusively in neurons and endocrine cells essential in neurotransmitter release (73).To test the production of mature synaptic vesicles, SV2A protein expression was detected in regenerated axons of RGCs after hPMSCs co-culture.We observed several SV2A positive puncti throughout RGCs regenerated axons, with similar size and shape as previously described (74) (Figure 5A).Those images demonstrated the presence of mature synaptic vesicles along those axons, suggesting their ability to release neurotransmitters in both oxygen conditions.
VGSCs are responsible of the initiation and propagation of potentials in excitable cells.VGSCs Nav1.1 α subunit (SCN1A) is expressed predominantly in cell bodies and dendrites and participate in generation of both somatodendritic and axonal action potentials (75,76) Immunostaining of SCN1A in hPMSCs-induced regenerated RGCs axons revealed in both conditions the presence of VGSCs in RGCs bodies, dendrites, and initial part of the axons (co-stained with MAP2A,B) suggesting their ability to generate action potentials.(Figure 5B) Taken together, these results confirm that RGCs axons, regenerated by hPMSCs induction, may have developed the subcellular and molecular capacity to be fully functional.
Once the neuroregenerative potential of hPMSCs and the expression of VGSC were confirmed, we proceeded to functionally explore the electrical activity of the regenerated neurons.For this purpose, we recorded the ionic currents generated by the cells maintained in normoxic and hypoxic conditions, by using the patch clamp technique, in "whole cell" configuration.
In a series of voltage clamping experiments carried out in regenerated cells, the ionic currents activated by cell depolarization were recorded.Depolarizing pulses were applied from a membrane potential of -80 mV in 5 mV step.Figure 6 shows the depolarization-activated currents recorded in a normoxic condition sample (Figure 6A), as well as in a hypoxic condition sample (Figure 6B).In both conditions, it is possible to observe the transient sodium current (INa), followed by the sustained potassium current (IK).These experiments, carried out in a group of regenerated axons in normoxia (n=8) and regenerated axons in hypoxia (n=7), allowed us to observe that the amplitude of the sodium currents recorded from regenerated cells in hypoxia was significantly lower than the current from the normoxic regenerated cells (Figure 6C).Similarly, the amplitude of potassium currents showed less amplitude in the hypoxic group than in the normoxic group (Figure 6D).Despite the observed differences, both cell types were able to generate action potentials when recorded in current clamp mode (Figure 6E,F).Therefore, our functional experiments demonstrate that the regenerated cells are active from an electrophysiological point of view.

DISCUSSION
Cell therapy is a state-of-the-art medical paradigm that encompasses the utilization of cellular entities to address diverse pathological conditions.It capitalizes on the exceptional regenerative and reparative proficiencies intrinsic to cells, facilitating the reestablishment or substitution of impaired tissues and fostering the recuperative process.Cellular therapy represents a paradigm-shifting prospect poised to redefine the healthcare landscape covering a spectrum of disorders including those affecting the nervous system.This pioneering domain encapsulates diverse modalities of cellmediated interventions, prominently featuring stem cell therapy (58,(77)(78)(79)(80)(81).
Stem cell-based regenerative medicine is increasingly seen as a compelling approach for developing therapies.Among the most coveted cell sources, MSCs stand out due to their distinctive attributes.MSCs orchestrate tissue development, maintenance, and repair, and are useful for regenerative therapies to treat degenerative diseases and other clinical conditions (82)(83)(84)(85).
Placenta-derived mesenchymal cells (hPMSCs) are a notable MSC source.They offer accessibility without invasiveness, possess strong immunomodulatory properties, high proliferative and differentiation potential, have the ability to migrate to injury sites in vivo and are ethically sound and versatile.For all these reasons, hPMSCs hold promise for regenerative medicine and better patient outcomes (59,(86)(87)(88)(89).
Recognizing the significant potential of these cells as therapy, our research group has directed its efforts towards further exploration and enhanced understanding of hPMSCs' potential as cellular strategy to achieve neuronal regeneration.
As a preliminary and crucial step in the study we ensured that the properties of the cells in culture remained consistent across passages.Ensuring that cells maintain their intended characteristics over time and culture conditions is crucial for the reliability and reproducibility of experiments and future therapeutic interventions.
The cells, maintained in culture from passages 2-8, grew adherent to plate and exhibited a typical spindle-shaped morphology as observed under inverted microscopy.No observable alterations in cell morphology were detected by optical microscopy.Following the examination of pluripotency markers (NANOG, Oct-4, and SOX2), it became apparent that the cells preserved their pluripotent characteristics.An immunophenotypic evaluation using flow cytometry assay was performed to evaluate the expression of mesenchymal and hematopoietic markers.Results confirmed that the cells exhibited immunophenotypic characteristics consistent with those of a mesenchymal lineage when maintained in culture.Specifically, the cells were positive for the surface markers CD105, CD44, and CD73, while they were negative for CD14, CD34, CD19, CD45 and HLA-DR.The cellular differentiation capacity was also verified.In summary, our data confirm that the cells maintain their mesenchymal characteristics throughout the culture procedure and treatments (71).
In relation to both environments to which the hPMSCs were exposed, growth kinetics assays revealed that hypoxic cultures exhibited a markedly higher cumulative population doubling potential.In addition to that our results strongly suggest that hypoxia improves the differentiation potential of these cells.
After thoroughly and rigorously defining and characterizing our hPMSCs, we tackled the experiments to investigate the potential of these cells in the process of adult CNS axotomized neurons axonal regeneration and restoration of neuronal activity.To address this goal, we used an in vitro co-culture model, previously established by Dr. Moreno-Flores`s team.Our group considered applying a similar approach to track and characterize the potential of hPMSCs in an axonal regeneration process.Specifically, hPMSCs and axotomized 2-month-old rat retinal ganglion cells (RGCs) shared the culture plate for 96 hours and percentage of neurons with axon and mean axonal length per neuron were quantified (21).
With our experimental approach, we achieved success getting 5 to 13-fold increase in the percentage of neurons with regenerated axons respect to RGCs in PLL.Moreover, the mean axonal length increased 4 and 24 times under normoxic and hypoxic conditions respectively.This result settled a very significant and promising result.
Numerous researchers have focused their efforts on exploring potential therapeutic strategies rooted in stem cell therapy for addressing conditions linked to neuronal degeneration or dysfunction.Nonetheless, while the predominant approach in these studies revolves around assessing the differentiation capacity of MSCs to support damaged cells, they ultimately conclude that the observed effects are mediated through paracrine signaling.A prevailing consensus among research collectives underscores the critical role of stem cells in facilitating the regeneration of injured nerves, primarily through the secretion of neurotrophic factors, including BDNF, NGF, NT-3 (23,59,61,79,80,(90)(91)(92)(93)(94)(95).
In line with previous studies, we confirmed that hPMSCs express the neurotrophic factors BDNF, NT3 and NGF.The western-blot results referred to the pro-factors expression indicate that pro-BDNF and pro NT-3 expression level were homogeneous along time in culture and with the different oxygen concentrations.However, the expression of the pro-NGF increased markedly when the cells were maintained in hypoxia.
Since the pro-factors should be cleaved intracellularly or extracellularly to generate their mature forms, we wanted to determine BDNF and NGF present in the conditioned media.It has been widely proven the role of BDNF as a key mediator in axonal regeneration by stimulating axonal growth and facilitating synaptic reorganization.It is demonstrated the role of BDNF in axonal regeneration of RGCs in co-culture with primary and immortalized rat OEG (23).In our system, we have found that hPMSCs (5x10 4 -10 5 ) release BDNF in concentration between 25±4.65 and 32±2.66 pg/ml with no difference when we compared secretion by cells maintained in normoxia and hypoxia conditions.NGF plays a crucial role in promoting the growth and survival of sensory neurons.Both the expression and the secretion of this factor appear to be hypoxia dependent.We have observed a twofold increase when comparing cultures maintained under hypoxia respect to normoxia.
It was of great interest to us to verify whether the RCGs regenerative effect of hPMSCs was due to a cell-to-cell contact, proximity-derived or a paracrine effect.To determine whether the previously tested effect was necessarily linked to a cell-cell contact we conducted indirect co-culture experiments, and secretome activity assays.The percentage of neurons exhibiting axonal regeneration in presence of conditioned media would quantify the contribution to this regeneration attributable to a neurotrophic impact of the hPMSCs.Furthermore, the mean axonal length per neuron provides an indicator for assessing the extent of axonal growth resulting from that neurotrophic influence.In addition to identified neurotrophic factors, other uncharacterized molecules secreted into the media may be playing a role in these regenerative parameters.
Significantly, in this experimental framework, we observed a reduction in the number of neurons exhibiting regenerated axons compared to the co-culture experiments involving RGCs and hPMSCs contact.Indeed, the number of regenerated RGCs closely mirrored the results obtained in RGC cultures without hPMSCs.Nevertheless, the paracrine impact of hPMSCs on axon length per neuron, under both normoxic and hypoxic conditions, exhibited an approximately three to fourfold increase when compared to the control RGCs cultured on PLL plates.These results suggest that hPMSCs play a key role as biological substrate strengthening the capacity of RGCs to regenerate their axons, partly through a paracrine mechanism.Additionally, we have shown that capacity to promote the highest axonal regeneration in this experimental paradigm depends on contact between the adult RGCs and the hPSMCs, as demonstrated for other regenerative cells, such as OEGs (27,90,96).
We also wanted to confirm the regenerated RGCs functionality.Synaptic vesicles were localized distributed along the regenerated axons, suggesting their capacity to release neurotransmitters.In addition to that we assessed the regenerated axons' ability to generate action potentials by characterizing the presence and activity of voltage-gated sodium channels (VGSCs).VGSCs play a pivotal role in the initiation and propagation of action potentials in neurons.Our findings demonstrate that the density of these channels is notably concentrated in the axon initial segment, a specialized region where action potentials are significantly greater than in the soma contributing to the distinctive electrical properties of this region.Based on our results, we can affirm that RGCs possess the molecular framework required for functionality.
Voltage-clamp experiments are a common method in neuroscience to measure the ionic currents in neurons.By controlling the membrane potential of the neuron, it is possible to measure the flow of ions in and out of the neuron during depolarization, providing valuable information about the neuron's function and health (97).
In our experiments, we have confirmed the expression of VGSCs in the regenerated neurons.Moreover, we proceeded to compare the ionic currents generated by cells maintained in normoxia or hypoxia culture.Our findings demonstrate that regenerated neurons, under both environmental conditions, manifest transient sodium currents (INa) and sustained potassium currents (IK) upon depolarization.Nevertheless, the amplitude of both sodium and potassium currents was notably reduced in the hypoxia-regenerated cells compared to those maintained under normoxic conditions, which could be due to the number and/or type of channels.In spite of these disparities, both cell types exhibited the capability to initiate action potentials, thereby affirming the electrophysiological activity of the regenerated cells representing without a doubt a biological breakthrough.
The study of the remarkably properties of hPMSCs, and the comprehensive understanding of all the molecules contributing to their ability to foster adult axonal regeneration undoubtedly warrant further investigation and approaches.by Oil Red O staining of lipids vacuoles.(G) Alcian Blue staining of proteoglycans demonstrated chondrogenesis differentiation.Scale bar: 100m.

Figure 6 .Figure 6
Figure 6.Study of electrical properties of regenerated axons.Axotomized adult retinal neurons were co-cultured with 8x10 4 hPMSCs/well and recorded under voltage/current clamp conditions.(A,B) Representative whole cell current recordings from cultured cells in response to voltage pulses of 5 mV increasing step from a holding voltage of -80 mV in conditions of normoxia (A) and hypoxia (B).(C,D) Current voltage relationship of the sodium currents (C) and potassium currents (D) averaged from a total of 8 cells in normoxic conditions and 7 cells in hypoxic conditions.(E,F) Representative whole cell voltage recordings from cells shown in A and B in response to a current pulse of 0,1 nA.